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Biomedical subjects

S B Horowitz

Publications and source records attributed to S B Horowitz.

At least 19 recordsLinked to original sources
PubMed1989

Gonadotropin stimulates oocyte translation by increasing magnesium activity through intracellular potassium-magnesium exchange.

We previously showed that gonadotropin increases the K+ activity in Xenopus oocytes and that this is a signal for increased translation. However, K+ need not act to control synthesis directly but may act through an unidentified downstream effector. Using microinjection to vary the salt content of oocytes and concomitantly measuring [3H]leucine incorporation, we found that small changes in Mg2+ greatly affect translation rates. (Ca2+ had little influence.) By measuring intracellular ion activities, we found that oocyte cations existed in a buffer-like (ion-exchange) equilibrium in which K+ and Mg2+ are the preponderant monovalent and divalent cations. Hence, increasing cellular K+ activity might increase translation by causing Mg2+ activity to rise. If so, the increased translation rates produced by hormone treatment or K+ injection would be prevented by EDTA, a Mg2+ chelating agent. This prediction was tested and confirmed. We conclude that, when gonadotropin increases K+ activity, the cell's internal ion-exchange equilibrium is altered thereby increasing Mg2+ activity and this up-regulates translation.

Animals↗
PubMed1988

Potassium salt microinjection into Xenopus oocytes mimics gonadotropin treatment.

Gonadotropin stimulates protein synthesis and growth in ovarian oocytes. The hormone is also known to modify transfollicular K+ fluxes and is now shown to cause increased intraoocytic K+ activity (aK). The hormone's effect on aK was duplicated by microinjecting K+ salts into oocytes which were incubated in paraffin oil. This treatment mimicked the influence of gonadotropin on both the rate of protein synthesis and the synthesis of specific polypeptides. These findings suggest that gonadotropin-stimulated oocyte growth is attributable largely to the hormone's influence on transfollicular K+ fluxes. They support the hypothesis that the K+ flux and aK changes observed during cell activation are critical in causing subsequent increases in protein synthesis and growth.

Animals↗
PubMed1988

A function that relates protein synthetic rates to potassium activity in vivo.

A newly developed experimental system allows the controlled alteration of intracellular K+ activity (aK) and the measurement of amino acid incorporation rates in a single cell, the Xenopus oocyte. We found that as aK is increased by microinjecting a K+ salt, [3H]leucine incorporation (R) varies over a 100-fold range, first stimulated and then inhibited as it passes through four response regions (A-D). In region A (aK approximately 60-100 mM), R is at a nongrowth or maintenance level and is stimulated weakly by increasing aK. In region B (aK approximately 100-130 mM), R is stimulated intensely by increasing aK, roughly tripling with every 10 mM increase. In region C (aK approximately 130-160 mM), R is inhibited intensely by increasing aK. Finally, in region D (aK greater than 160 mM), R is inhibited weakly as aK increases. Collectively, the four response regions constitute the oocyte's R/aK response function. The function provides a comprehensive description of how K+ activity influences the rate of protein synthesis in an intact cell. In the subsequent discussion, we compared the oocyte response function with the K+ response determined in cell-free translational systems. While in vivo and in vitro functions are similar, differences exist that may be important in a cellular control system. We then considered the relevance of the oocyte R/aK response function to "normal" processes in the oocyte and in somatic cells, i.e., those in which aK is varied by physiological changes in the plasma membrane. We concluded that the intensely stimulatory region B is importantly involved in hormonal action and other growth-activating processes and that the entire R/aK response function may play a role in control of protein synthesis during the cell cycle.

Animals↗
PubMed1986

Intracellular compartmentalization of adenosine triphosphate.

The intracellular distribution and diffusivity of adenosine triphosphate (ATP) was studied by cryomicrodissection of individual Rana pipiens oocytes. We measured ATP concentrations in the nucleus, in animal and vegetal hemisphere cytoplasm, and in an intracellular reference phase (iRP, a microinjected gelatin "organelle") which samples diffusive ATP. Regional concentrations were not equal: nucleus much greater than animal ooplasm greater than vegetal ooplasm. ATP binding and water availability (as solvent) were determined by plotting nuclear and cytoplasmic ATP concentrations as a function of reference phase ATP concentrations (isothermal analysis). The nucleus/iRP isotherm for ATP was an equimolar line, showing that nucleoplasm resembles iRP gelatin (and consequently a simple aqueous solution) in its solvent properties. Cytoplasm/iRP isotherms were more complex, having slopes much less than unity and ordinal intercepts above the graph's origin. They demonstrate the presence in cytoplasm of mechanisms that are capable of excluding and binding ATP. These mechanisms are responsible for the inhomogeneity in ATPs intracellular distribution. In addition, exclusion and binding have different and opposing effects on ATP concentrations in the cell's "soluble space," and hence on ATP availability to enter into cellular reactions. It follows that these phenomena must be considered in attempts to model ATPs role in metabolism.

Adenosine Triphosphate↗
PubMed1984

Water, potassium, and sodium during amphibian oocyte development.

Water, K+, and Na+ were measured in Rana pipiens oocytes during growth and prematurational development using low-temperature microdissection. Whole oocytes were analyzed during previtellogenic and vitellogenic growth. Ooplasm and germinal vesicle (nucleus) were analyzed at the onset and conclusion of vitellogenic growth. In previtellogenic oocytes (less than 40 micrograms), water, K+, and Na+ concentrations resembled those in somatic cells and were independent of cell size. With the onset of yolk deposition, water and K+ concentrations progressively decreased and Na+ progressively increased. These changes were restricted to ooplasm, the site of yolk deposition. In full-grown oocytes, vegetal ooplasm, with greater yolk density than animal ooplasm, contained less water and K+ and more Na+ than animal ooplasm. Collectively, the data indicate that yolk is poorer in water and K+ and richer in Na+ than yolk-free ooplasm (cytoplasm) or nucleoplasm. Yolk concentrations were estimated to be approximately 32%, water, approximately 69 meq K+/liter H2O, and approximately 94 meq Na+/liter H2O. Several nonyolk parameters, such as cation activities and nucleoplasmic binding, also appear to change during oogenesis.

Animals↗
PubMed1984

Regional water changes during oocyte meiotic maturation: evidence of ooplasmic segregation.

Cryomicrodissection was used to measure the intraoocytic distribution of water before and during meiotic maturation in Rana pipiens oocytes. Animal ooplasm contained about 10% more water in matured than in ovarian oocytes. The increase was not dependent on the uptake of extracellular water, occurring even when oocytes were matured in a paraffin oil medium. Rather, animal ooplasm hydration appeared to be due to an increase in the volume fraction occupied by cytoplasm (reduced yolk density) through: (1) migration of cytoplasm from the vegetal to animal hemisphere and (2) mixing of ooplasm with nuclear sap during germinal vesicle breakdown (GVBD). Cytoplasmic migration (or ooplasmic segregation) began prior to GVBD, probably within an hour of exposure to progesterone and appeared to continue through the period of GVBD. The volume of cytoplasm that moved significantly reduced water concentrations in vegetal ooplasm at 6 hr postprogesterone and offset any subsequent water gain due to the mixing of nuclear sap and vegetal ooplasm at GVBD. The findings suggest that segregational movements are among the early maturational changes entrained by progesterone. Ooplasmic segregation is considered in the context of theories of cytomatrix movement in which control resides in regional Ca2+ activity gradients. We address the problem of the vegetal----animal directionality of movement and suggest that the annulate lamellae play a role.

Animals↗
PubMed1984

Artifacts caused by cell microinjection.

The effects of microinjection on Rana pipiens oocytes were determined using cryomicrodissection to measure Na, K, water, and injected radiolabeled sucrose (in gelatin) in the nucleus, animal, and vegetal ooplasm and injected bolus (reference phase, RP). The results point to potential problems in the interpretation of microinjection experiments. When oocytes were injected and incubated in Ringer's solution, nucleus, ooplasm, and RP lost K and sucrose and gained Na. Patterns of loss and gain were complex but were consistent with continuous solute leakage at the injection site causing artifactual intracellular diffusion gradients. In spite of leakage, oocytes completed scheduled meiotic maturation when exposed to progesterone. When oocytes were microinjected and incubated in paraffin oil (a medium in which polar solutes cannot exchange), nuclear and ooplasmic Na, K, and water concentrations remained identical to those in uninjected cells. Neither microinjection per se nor the injected bolus affected intraoocytic solute distributions. These findings imply that, after microinjection in aqueous media, metabolites are lost from and redistribute in cells, and that these artifactual changes are inadequately reflected in the ability of the cell to carry out a complex process. They also show that injection artifacts can be avoided by injecting and incubating cells under paraffin oil.

Animals↗
PubMed1984

Solvent properties of ground substance studied by cryomicrodissection and intracellular reference-phase techniques.

Water, sodium, potassium, ATP, amino acids, and sugars are not uniformly distributed in Rana pipiens oocytes. Concentration differences exist between nucleus (germinal vesicle) and ooplasm and between animal and vegetal ooplasmic regions. The mechanisms responsible for these differences were investigated using intracellular reference-phase (iRP) analysis. The iRP is an artificial "organelle" that has the solvent properties of a dilute salt solution and is in diffusional equilibrium with water and solutes present in other cellular compartments. Ooplasm/iRP solute distributions show that ooplasm differs from ordinary aqueous solutions--exhibiting both solute exclusion and solute binding. Yolk platelets are an important cause of this behavior, largely because their proteins are present as hydrate crystals, which are rich in anionic sites and which interact intensely with associated water. Because of yolk's abundance, it obscures the solvent and binding properties of ooplasmic ground substance. The oocyte nucleus is yolk and organelle free and the nuclear envelope is readily permeable. Consequently, nucleus/iRP solute concentration differences reflect the binding and solvent properties of nuclear ground substance. Nucleoplasm binds approximately 19 meq of potassium. Furthermore, the monosaccharides, 3-O-methylglucose, L-glucose, and D-xylose, are selectively excluded, their nucleus/iRP concentration ratios averaging about 0.7; ratios for other solutes studied are unity. We interpret monosaccharide exclusion to mean that nuclear ground substance water is different in its "instantaneous" structure from ordinary saline water. Because of this difference, hydrogen bond interaction between nuclear water and certain sterically restricted solutes, of which ringed monosaccharides are examples, is reduced. Some implications of modified ground substance water and selective solute exclusion are discussed.

3-O-Methylglucose↗
PubMed1983

Protein loss during nuclear isolation.

Cryomicrodissection makes possible the measurement of the entire in vivo protein content of the amphibian oocyte nucleus and provides a heretofore missing baseline for estimating protein loss during nuclear isolation by other methods. When oocyte nuclei are isolated into an aqueous medium, they lose 95% of their protein with a half-time of 250 s. This result implies an even more rapid loss of protein from aqueously isolated nuclei of ordinary-size cells.

Animals↗
PubMed1980

Potassium exchange in the whole cell, cytoplasm, and nucleus of amphibian oocytes.

Potassium isotope exchange was studied in whole oocytes, with and without ovarian follicles, and in oocyte cytoplasm and nucleus. Cryomicrodissection was used to prevent solute redistribution during nuclear and cytoplasmic separation. Manual follicle removal causes a small decrease in the K+ of the preparation. No effect of follicle removal is seen on 42K+ exchange. Whole oocyte exchange is multiphasic and reflects the presence of two intracellular K+ fractions. One of these fractions is present in both nucleus and cytoplasm. It exhibits first-order exponential kinetics, apparently established at the cell membrane. The second fraction is restricted to cytoplasm and exchanges at an imperceptible rate. The fractions differ in pre- and posthibernation oocytes. These observations clarify the mechanism whereby nuclear/cytoplasmic K+ concentration asymmetries are maintained and the mechanisms responsible for the high K+-activity coefficient previously reported in these cells.

Animals↗
PubMed1979

Reference phase analysis of free and bound intracellular solutes. I. Sodium and potassium in amphibian oocytes.

A method is described for the quantitative determination of free and bound solute concentrations in the cytoplasm of intact cells. The method includes (a) introduction of a gelatin gel reference phase (RP) into the cytoplasm; (b) diffusion of dissolved substances between cytoplasm and RP, (c) cell quenching to - 196 degrees C to prevent subsequent solute redistributions, (d) ultra-low temperature microdissection to isolate RP and cytoplasm samples, and (e) analysis of isolates for solute and water content. In normal oocytes of the salamander, Desmognathus ochrophaeus, free or RP Na+ and K+ are 21.0 +/- 1.1 and 128.8 +/- 2.4 mu eq/ml, respectively, and vary stoichiometrically in altered oocytes. Overall cytoplasmic concentrations are 75.2 +/- 2.7 mu eq Na+/ml and 88.6 +/- 1.5 mu eq K+/ml. Cytoplasmic chemical activities are 16.2 mu eq Na+/ml and 99.2 mu eq K+/ml, corresponding to activity coefficients of 0.22 and 1.12, respectively. The results demonstrate unambiguously that (a) oocytes actively transport Na+ and K+, and (b) cytoplasm has important binding properties which differentiate it from an ordinary aqueous solution. These cytoplasmic properties are investigated in the following paper.

Animals↗
PubMed1979

Reference phase analysis of free and bound intracellular solutes. II. Isothermal and isotopic studies of cytoplasmic sodium, potassium, and water.

The intracellular reference phase (RP) method and ultra-low temperature micro-dissection were used for isothermal and isotopic phase distribution studies of Na(+), K(+), and water in amphibian oocyte cytoplasm. One-third of the cytoplasmic water is available as solvent for [(3)H]sucrose. This fraction, designated c1, quantitatively coincides with the water volume in which Na(+) and K(+) are freely diffusible. Two-thirds of the cytoplasmic water is inaccessible to sucrose and is designated c2. The Na(+) and K(+) associated with c2 are extremely slowly exchanging (bound) and at different concentrations than in c1. The cations in c1 are in mass-action equilibria with those in c2, each described by an equation of the formC(c) (i) = C(c) (1) (i) + C(c) (2) (i) = q(i).C(RP) (i) + (max)C(c) (2) (i).f(C(RP) (i)in which C(c) (i) is the cytoplasmic Na(+) or K(+) concentration, C(c) (1) (i) is the free, and C(c) (2) (i) the bound cation concentration averaged over the cytoplasmic water. q(i) is the fractional free solute space, C(RP) (i) the RP concentration, (max)C(c) (2) (i) the concentration of binding sites, and the function f is satisfied by the Langmuir isotherm. Numerical values for the variables of the isotherm are determined. Activity coefficients are calculated from RP data and provide a basis for generalizing the oocyte results to other cells. The conclusion is drawn that both c1 and c2 are widely distributed in cells, and that cellular ionic activities involve two distinct systems: the cell-membrane system and an adsorbed water ion-exchange-like buffering system. Alternative explanations for the two-component cytoplasm are considered. A model is proposed in which c1 is a normal intracellular aqueous phase controlled by the plasma membrane, whereas c2 consists of water and ions adsorbed in hydrate crystalline structures. In oocytes these structures are identified with yolk platelets.

Animals↗
PubMed1975

Nuclear envelope permeability.

The permeability of the amphibian oocyte nuclear envelope in situ has been determined for three tritiated dextrans. The envelope is a sieve, restricting molecular movement between the cytoplasm and nucleus. The patent radius of its pores is about 45å.

Amphibians↗
PubMed1975

Nucleocytoplasmic transport and distribution of an amino acid, in situ.

Ultra-low temperature techniques (microdissection and autoradiography) were used to study the nucleocytoplasmic distribution and transport of alpha-aminoisobutyric acid (AIB) in an amino acid-accumulating cell. In amphibiam oocytes incubated in AIB, the nuclear concentration of this non-metabolizable amino acid exceeds the cytoplasmic concentration by 45%, remaining constant both over time and variation in substrate concentration. The kinetics of uptake suggest that this nucleo-cytoplasmic asymmetry arises from solubility differences between the 2 compartments, and that the nuclear envelope plays a negligible role in amino acid transport. A solute exclusion model is offered to explain the nucleocytoplasmic asymmetry.

Aminoisobutyric Acids↗